Synthetic Molecules

Courtesy Photo | Methodology used to compare 120 nucleotide-maximum oligos produced enzymatically (Syntax-100, DNA Script, Inc.) to chemically synthesized 100 nucleotide-maximum oligos (Integrated DNA Technologies) in gene assembly. (DEVCOM CBC image)  

FORT BELVOIR, VA, UNITED STATES

01.06.2024

Courtesy Story

Defense Threat Reduction Agency's Chemical and Biological Technologies Department

Benchmarking the platforms to improve DNA synthesis

Advancements in DNA synthesis technologies have the potential to lower some of the technical and costly barriers that scientists have faced since the discovery of the DNA molecular function. DNA has become a valuable tool for forensics, chemical, and biological sciences capable of generating biological systems, including organisms, from synthetic genomes. Synthetic biology is an area where technological advances affect the threat landscape that will ultimately influence chemical and biological (CB) defense to protect warfighters from current and emerging CB threats. These new advancements include creating synthetic DNA faster.

To take advantage of the significant advancements in DNA synthesis that have occurred over the past five to ten years, the Defense Threat Reduction Agency’s (DTRA) Chemical and Biological Technologies Department in its role as the Joint Science and Technologies Office (JSTO) for Chemical and Biological Defense, an integral component of the Chemical and Biological Defense Program, invested with the U.S. Army Combat Capabilities Development Command Chemical Biological Center (DEVCOM CBC) to assess new enzymatic methodologies in terms of technical advantages, costs benefits, and an overall decrease of technical barriers.

The DEVCOM CBC team evaluated commercial enzymatic DNA synthesis (EDS) printers as they offer a user-friendly platform and predominantly fluid waste stream compared to the traditional large amounts of waste generated by chemical DNA synthesis (CDS). Scientists showed that a benchtop EDS can produce a 96-well plate of 60-nucleotide-long oligonucleotides (“oligos”) in 13 hours, including cleanup and quantitation (the average concentrations of DNA present in a mixture, as well as their purity). In contrast, an equivalent run on a benchtop CDS platform requires two days for synthesis, cleanup, and quantitation. Commercial oligos then typically require a two-day delivery. Notably, the EDS instrument produced only 1 liter of primarily aqueous, non-hazardous waste compared to 8 liters of hazardous organic waste for a similar run on a benchtop CDS platform.

The researchers also evaluated enzymatically produced oligos alongside chemically produced commercial oligos to assemble a synthetic gene-encoding Green Fluorescent Protein (GFP). The EDS platform produced lower concentrations of individual oligos, but these were available in half the time of commercially produced oligos and were sufficient to assemble functional GFP sequences of comparable accuracy without producing hazardous chemical waste.

Currently, phosphoramidite-based CDS is the standard method for creating new single-strand DNA building blocks and is used extensively by commercial vendors. The advances in EDS make this technology competitive with chemical methods, with the potential of shifting to large-scale enzymatic DNA synthesis in the near future.

Overall, benchtop EDS platforms can produce more oligos in a shorter time frame at a comparable cost to similar CDS instruments while offering a convenient, user-friendly interface with control over final yield, sequence-editing capabilities, and an environmentally friendly waste stream. The knowledge and experience required to use these benchtop enzymatic DNA platforms is minimal and significantly lowers the barriers for use across many disciplines and applications. However, only one company currently sells a benchtop EDS instrument, which increases the overall cost of the instruments and reagents. Additionally, the current benchtop EDS platform is less amenable to manual user input throughout the synthesis compared to CDS instruments and requires skilled personnel to address instrument malfunctions or errors. Nevertheless, multiple companies have made strides in using EDS methods to overcome the 100-oligo length limitation faced by chemical methods by producing single oligos up to 1000 nucleotides long.

With such advances, enzymatically produced DNA methods will continue to evolve, substantially changing the biological threat landscape with the production of longer DNA sequences for future applications to protect the Joint Force.

POC: Giselle Roman Hernandez, Ph.D., [e-mail]

• Date Taken: 01.06.2024
• Date Posted: 01.07.2024 00:17
• Story ID: 461417
• Location: FORT BELVOIR, VA, US